Kidney Diseases And Disorders. The kidney functions to separate waste products from the blood, regulate acid concentration, and maintain water balance. Kidneys control the levels of various compounds in the blood, such as hydrogen, sodium, potassium, and silicon, and eliminate waste in the form of urine. Any degradation in kidney function can interfere with the body's ability to adequately remove metabolic products from the blood, and can disrupt the body's electrolyte balance. In its most severe forms, degradation or impairment of kidney function can be fatal.
A number of conditions can lead to chronic renal failure, a decline in kidney function over time. For example, such conditions as hypertension, diabetes, congestive heart failure, lupus, and sickle cell anemia have been associated with renal failure. Acute disease processes and injuries can trigger a more immediate decline in kidney function.
It is thus well understood that individuals with diabetes, hypertension, inflammatory and autoimmune diseases, and other disorders are at risk for altered and progressive loss of kidney function characterized by, for example, reduced glomerular filtration, albuminuria, proteinuria, and progressive renal insufficiency. More than half of the total number of kidney disorders initiate kidney fibrosis. Fibrosis involves altered formation or production of fibrous tissue, and can result in the overproduction and increased deposition of extracellular matrix components.
The extracellular matrix (ECM) is a complex network of various glycoproteins, polysaccharides, and other macromolecules secreted from a cell into extracellular space. The ECM provides a supportive framework, directly influencing various cellular characteristics, including shape, motility, strength, flexibility, and adhesion. In fibrosis, overproduction and increased deposition of ECM materials can result in thickening and malformation of various membranous and cellular components, reducing local flexibility and surface area of the affected site, and impairing a number of bodily processes.
Kidney fibrosis is a common pathway in the progression of various forms of renal injury. Kidney fibrosis typically spreads by enlisting previously undamaged regions of the kidney. As normal filtration processes decline, function of surviving tissue and of various regions of the kidney is systematically destroyed. Kidney fibrosis can be manifested as a diffuse thickening of kidney membranous components, the accumulation and expansion leading to a loss of filtration surface area and a corresponding disruption in the body's electrolyte composition and acid-base balance.
Fibrosis of the kidney is observed in a number of conditions, including, for example, diabetic, autoimmune, and transplant nephropathy; hypertension; and certain forms of glomerular injury or disease. Diabetes mellitus (diabetes) is a complex disease that affects several hundred million people worldwide. Diabetes is characterized by hyperglycemia or elevated levels of glucose in the blood. Glucose cannot enter the body's cells to be utilized and therefore remains in the blood in high concentrations. When the blood glucose level exceeds the reabsorptive capacity of the renal tubules, glucose is excreted in the urine. Diabetes produces a number of debilitating and life-threatening complications.
Progressive nephropathy is one of the most frequent and serious complications of diabetes. See, e.g., Hans-Henrik et al., 1988, Diabetic Nephropathy: The Second World Conference on Diabetes Research, New Frontiers. The Juvenile Diabetes Foundation International, pp. 28-33. A hallmark of diabetic nephropathy, and of renal sclerosis due to other forms of renal injury, is early expansion of the glomerular mesangium, largely due to increased accumulation of ECM proteins such as collagen types I and IV, fibronectin, and laminin. See, e.g., Mauer et al., 1984, J Clin Invest 74:1143-1155; Bruneval et al., 1985, Human Pathol 16:477-484. This pathological deposition results in impaired filtration, leading to renal failure, a condition requiring transplantation or life-long dialysis. Current therapies slow but do not arrest or reverse the progressive loss of kidney function. Predominant causal factors identified to date also include hyperglycemia, glomerular hypertension, and abnormal cytokine environments. Tuttle, et al., 1991, N Engl J Med 324:1626-1632; The Diabetes Control Complications Trial Research Group, 1993, N Engl J Med 329:977-986; Hostetter et al., 1981, Kidney Int 19:410-415; Anderson et al., 1985, J Clin. Invest 76; 612-619; Border et al., 1993, Am J Kidney Dis 22:105-113.
Hyperglycemia may be damaging, in great part as increased concentrations of glucose stimulate ECM accumulation by mesangial cells. See, e.g., Ayo et al., 1990, Am. J. Pathol. 136:1339-1348; Heneda et al., 1991, Diabetologia 34:190-200; Nahman et al., 1992, Kidney Int 41:396-402; Cortes et al., 1997, Kidney Int. 51:57-68. As shown by Davies et al., 1992, Kidney Intl. 41:671-678, mesangial cells are largely responsible for mesangial matrix synthesis in situ. It has further been determined that the effect of glucose on mesangial cell matrix production is linked to increased glucose transport and utilization. Helig et al., 1995, J. Clin. Invest. 96:1802-1814. Moreover, Ziyadeh et al., 1994, J. Clin Invest. 93:536-542, have shown the involvement of secreted soluble mediators on mesangial cell matrix production.
Renal hypertension, which can appear as a secondary manifestation of kidney disease in diabetic patients, can also result from other diseases or disorders, including long-standing hypertension. Secondary hypertension can be caused by virtually any impairment in renal function. A greater understanding of the pathogenic mechanisms for hypertension-induced ECM deposition is developing. For example, in diabetes, an early impairment of normal blood pressure dampening occurs at the glomerular afferent arteriole, resulting in the exposure of glomerular capillaries to large moment-to-moment variations in systemic blood pressure. Hayashi et al., 1992, J Am Soc Nephrol 2:1578-1586; Bidani et al., 1993, Am J Physiol 265:F391-F398. Due to the elasticity of the glomerulus, increased capillary pressure produces expansion of glomerular structure, resulting in augmentation of the mechanical strain imposed on the mesangial cells. Riser et al., 1992, J Clin Invest 90:1932-1943; Kriz et al., Kidney Int Suppl 30:S2-S9. In addition, when cultured mesangial cells are subjected to cyclic strain, the mesangial cells respond by increasing the synthesis and accumulation of collagen types I and IV, fibronectin, and laminin. Riser et al., 1992, supra. While increased glomerular pressure is common in diabetes, it is not limited to this disease, and is present in other forms of progressive renal disorders, including, for example, certain forms of glomerular nephritis and hypertrophy. See, e.g., Cortes et al., 1997, Kidney Int 51:57-68.
Kidney fibrosis and associated renal impairment are thus present in the progression of various diseases and disorders, including diabetes and hypertension, and methods of treating kidney fibrosis are thus greatly desired.
Transforming Growth Factor β (TGF-β). The few studies conducted to date regarding the physiological implications of renal disorders and diseases, and, in particular, those due to diabetes, have focused on the role of transforming growth factor-β (TGF-β) in developing methods for targeting overproduction (increased synthesis and accumulation) of extracellular matrix components. The role of cytokine imbalance in initiating and/or perpetuating glomerular matrix expansion has been explored in experimental nephropathy studies involving TGF-β. See, e.g., Sharma et al., Seminars In Nephrology 1: 116-129. Glomerular TGF-β activity is increased in both human and experimental diabetic glomerulosclerosis. See, e.g., Yamamoto et al., 1993, Proc Natl Acad Sci 90:1814-1818; Sharma et al., 1994, Am J Physiol 267:F1094-F1101; Shankland et al., 1994, Kidney Int 46:430-442. The exposure of cultured mesangial cells or glomeruli to TGF-β results in increased ECM production. See, e.g., Bollineni et al., 1993, Diabetes 42:1673-1677. In vivo induction of glomerular matrix accumulation following transfection and overexpression of the TGF-β gene in rat kidney has been demonstrated by, for example, Isaka et al., J Clin Invest 92:2597-2601.
In addition, neutralization studies have shown that anti-TGF-β antibody mitigates the enhanced glomerular ECM gene expression that occurs in experimental glomerulonephritis and diabetes. Border et al., 1990, Nature 346:371-374; Sharma et al., 1996, Diabetes 45:522-530. The sustained overexpression of glomerular TGF-β in diabetes may be the result of a mesangial cellular response to both increased glucose levels and hypertension. It has been reported that exposure of mesangial cells to increased concentrations of glucose in the medium stimulates the synthesis and release of TGF-β1, as well as the increased binding of TGF-β to specific receptors. Ziyadeh et al., 1994, J Clin Invest 93:536-542; Riser et al., 1998, J Am Soc Nephrol 9:827-836; Riser et al., 1999, Kidney Int 56:428-439. It has also been reported that mechanical force selectively stimulates the production, release, and activation of TGF-β1, as well as the increased expression of TGF-β receptors. Riser et al., 1996, Am J Path 148:1915-1923.
In vitro neutralization studies of TGF-β demonstrated a significant reduction of collagen synthesis induced in mesangial cells by increased glucose levels. See, e.g., Sharma et al., 1996, supra; Ziyadeh et al., 1994, supra. Studies have also shown a virtual elimination of collagen accumulation resulting from cyclic stretching in the presence of excess glucose. Riser et al., 1997, supra. TGF-β stimulates the proliferation of mesangial cells in vitro and in vivo, and may induce in these replicating cells overproduction and increased deposition of ECM characteristic of various renal disorders, including proliferative disorders such as glomerular nephritis. See, e.g., Border et al., 1990, Nature 346:371-374; Habershroh et al., 1993, Am J Physiol 264:F199-205. As a result of these findings, intense efforts have been directed toward reducing TGF-β availability and binding as a means of mitigating matrix accumulation. However, the ubiquitous nature and pluripotent functions of TGF-β, including tumor suppression and the multiple levels of regulation, raise questions concerning both the feasibility and the safety of its long-term inhibition. See, e.g., Brattain et al., 1996, Curr Opin Oncol 8:49-53; Franklin, 1997, Int J Biochem Cell Biol 29:79-89.
Therefore, a method for treating or preventing ECM overproduction or increased deposition, without interfering with the ubiquitous function of TGF-β, is needed.
Connective Tissue Growth Factor (CTGF). CTGF is a peptide that may act downstream of TGF-β to regulate matrix accumulation. This novel growth factor has been reported and described previously. See, e.g., U.S. Pat. No. 5,408,040; Bradham et al., 1991, J Cell Biol 114:1285-1294. CTGF is characterized as a polypeptide which exists as a monomer with a molecular weight of approximately 36 to 38 kD. CTGF has been shown to be one of seven cysteine-rich secreted proteins belonging to the CCN family, which includes CTGF, cyr-61, and nov. Oemar et al., 1997, Arterioscler Thromb Vasc Biol 17(8):1483-1489. CTGF is an immediate early response gene that codes for a protein consisting of four modules and one signal peptide. Oemar et al., 1997, supra. The four modules are: 1) an insulin-like growth factor (IGF) binding domain, 2) a von Willebrand factor type C repeat most likely involved in oligomerization, 3) a thrombospondin type 1 repeat believed to be involved in binding to the ECM, and 4) a C-terminal module which may be involved in receptor binding. Recent reports suggest that certain fragments of the whole CTGF protein possess CTGF activity. See, e.g., Brigstock, et al., 1997, J Biol Chem 272(32):20275-20282. Human, mouse, and rat CTGF are highly conserved with greater than 90% amino acid homology and a molecular weight of about 38 kD. It was recently shown that the promoter of CTGF contains a novel TGF-β responsive element. Grotendorst et al., 1996, Cell Growth Differ 7:469-480.
It appears that CTGF may be an important prosclerotic molecule in both skin fibrosis and cardiac atherosclerosis. For example, CTGF mRNA is expressed by fibroblasts in the lesions of patients with systemic sclerosis, keloids, and localized scleroderma, while there is no corresponding expression in adjacent normal skin. See, e.g., Igarashi et al., 1995, J Invest Dermatol 105:280-284; Igarashi et al., 1996, J Invest Dermatol 106:729-733. Cultured normal human skin fibroblasts respond to TGF-β but not to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF), by increasing levels of CTGF mRNA and CTGF protein. Igarashi et al., 1993, Mol Biol Cell 4:637-645. Fibroblasts from lesions of scleroderma show increased mitogenesis to TGF-β and produce greater amounts of CTGF than do normal fibroblasts. Kikuche et al., 1995, J Invest Dermatol 105:128-132. Recombinant human CTGF injected under the skin of NIH Swiss mice induces the same rapid and dramatic increase in connective tissue cells and ECM as occurs with TGF-β treatment, whereas PDGF and EGF have little or no effect on granulation. Frazier et al., 1996, J Invest Dermatol 107:404-411. Cultured vascular smooth muscle cells are also stimulated by TGF-β to produce CTGF. In heart disease patients, CTGF mRNA is expressed at levels 50- to 100-fold higher in atherosclerotic plaques than in normal arteries. Oemar et al., 1997, Circulation 95(4):831-839.
In spite of mounting evidence implicating CTGF as a causal factor in skin fibrosis and cardiac atherosclerosis, very little is known of its expression in, for example, renal sclerosis or diabetes. It has been shown, using an in vitro model of calcium oxalate nephrolithasis, that monkey kidney epithelial cells respond to calcium oxalate by upregulating the CTGF gene along with other genes involved in matrix turnover. Hammes et al., 1995, Kidney Int 48:501-509. A similar response occurs in cultured renal epithelial cells following mechanical wounding. See, e.g., Pawar et al., 1995, J Cell Physiol 165:556-565. Most recently, CTGF mRNA was found in biopsies from normal human kidneys. A qualitative assessment indicated that, in a limited number of cases, CTGF expression was increased in the tissues of patients with severe mesangial proliferative lesions of crescentic glomerulonephritis, focal and segmented glomerulosclerosis, and, in three cases, diabetic glomerulosclerosis. Ito et al., 1998, Kidney Int 53:853-861. The research, relying only on data obtained from biopsies, did not include quantitative results or any measurement of CTGF protein levels. Further, no connection between CTGF mRNA levels and the production and deposition of ECM, and no quantitative method for detecting renal disorders or diseases, including diabetes, involving a determination of CTGF levels in samples, and did not identify CTGF-expressing cells.
The role of CTGF in kidney diseases is thus unclear, and there has been no research to date has shown that CTGF is causally related to ECM overproduction and increased deposition and to fibrosis in the kidney.
Diagnostics and Early-Stage Detection. Kidney failure is a serious condition requiring extreme treatment such as hemodialysis or transplantation. Early-stage detection and/or prevention of any deviation from normal kidney pathology and function could minimize the risk of a subject's developing a more serious condition. Hypertension, for example, might be undetectable by a patient in early stages, but can be deadly if not identified, monitored, and treated. In addition, in some diseases, such as, for example, diabetes, less invasive and disruptive and more affordable means of treatment, such as dietary modification, are effective only at early stages. Therefore, there is a critical need for effective and reliable methods of diagnosis that permit early stage detection, and corresponding prevention, of renal complications.
For example, kidney failure resulting from progressive glomerulosclerosis is the leading cause of morbidity and mortality among patients with type I, or juvenile, diabetes mellitus. See, e.g., Dorman et al., 1984, Diabetes 33:271-276; Anderson et al., 1983, Diabetologia 25:496-501. Current therapy with angiotensin-converting enzyme (ACE) inhibitors, the drug class of choice, effectively slows the progression of disease. See, e.g., Lewis et al., 1993, N Eng J Med 329:1456-1462. Nevertheless, this treatment is not justified in all newly diagnosed diabetic patients because only approximately 30-35% of these develop progressive kidney disease, and the long-term side effects of these drugs are uncertain. See, e.g., Parving and Hommel, 1989, Brit Med J 299:230-233. In addition, ACE inhibitors are also presently used to treat patients with hypertensive renal failure, including that resulting from non-diabetic nephropathies. However, the mechanism of renal protection, and, as noted above, the long-term side effects of this treatment are not fully understood. Furthermore, ACE inhibitors have been shown to negatively interact with nonsteroidal anti-inflammatory drugs. See, e.g., Whelton, 1999, Am J Med 106(SB):13S-24S.
In a current method of diagnosis, diabetic patients are monitored for microalbuminuria. Persistent microalbuminuria is a marker of widespread vascular damage and indicates the presence of early nephropathy in type 1 and type 2 diabetes. See, e.g., Stehouwer et al., 1992, Lancet 340:319-323; Bojestig et al., 1996, Diabetes Care 19:313-317; Mogensen et al., 1995, Lancet 346:1080-1084. However, the actual level of microalbuminuria may not necessarily predict the development of overt nephropathy, particularly among patients with a long duration of diabetes. Bojestig et al., supra. In addition, since by the time microalbuminuria is detected, structural renal lesions are already present, the effectiveness of treatment to slow progression may be substantially reduced. Bangstad et al., 1993, Diabetologia 36:523-529; Ruggenenti et al., 1998, J Am Soc Nephrol 9:2157-2169; Fioretto et al., 1995, Kidney Int 48:1929-1935. There is a great need to be able to predict which patients with type 1 diabetes will develop nephropathy, and to, in general, develop a method that will detect renal alterations that may precede the onset of significant disease.
In summary, there is a need in the art for effective methods for diagnosing, treating, and preventing fibrosis associated with impairment and degradation of kidney function in a variety or diseases and disorders, most particularly, in diabetes and hypertension. No current research has focused on the modulation of CTGF expression or activity as a means of preventing or treating kidney fibrosis.